Impact of Germination on Biochemical and Antioxidant Enzymes of Ceiba pentandra (Kapok) Seeds

doi:10.4236/ajps.2012.39144

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American Journal of Plant Sciences, 2012, 3, 1187-1192

http://dx.doi.org/10.4236/ajps.2012.39144 Published Online September 2012 (http://www.SciRP.org/journal/ajps)

1187

Impact of Germination on Biochemical and Antioxidant

Enzymes of Ceiba pentandra (Kapok) Seeds

Chekuboyina Ravi Kiran1*, Dadi Bhaskar Rao1, Nagala Sirisha1, Tamanam Raghava Rao1

1Department of Biochemistry, College of Science and Technology, Andhra University, Visakhapatnam, India.

Email: *[email protected]

Received March 29th, 2012; revised April 27th, 2012; accepted May 6th, 2012

ABSTRACT

Changes in biochemical components and the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase

(POD), glutathione peroxidase (GPx) and ascorbate oxidase (AO) in germinating and non-germinating seeds of Ceiba

pentandra were evaluated in the present study. Results show that the levels of proteins and total soluble sugars are high

and reducing sugars and free amino acids are low in non-germinating seeds whereas the contrary was observed in 4

days old germinating seeds. Enzymatic antioxidants like SOD, CAT, POD, GPx and AO showed enhanced activities

during seed germination. Our findings indicate that three days after germination of Ceiba pentandra seeds, their palat-

ability significantly improve. The nutritive utilization of protein and carbohydrates along with efficient participation of

antioxidant mechanisms, including the synergistic activities of the different types of SOD, CAT, POD, GPx and AO,

might play an important role during seed germination. The conclusion of this inquiry is that the germinating seeds can

serve as natural antioxidant agents, setting ahead the possibility of employing them for therapeutic purposes.

Keywords: Amino Acids; Ascorbate Oxidase; Catalase; Germination; Glutathione Peroxidase; Peroxidase; Reducing

Sugars; Superoxide Dismutase

1. Introduction

Seed germination and post-germination seedling deve-

lopment are well-regulated processes in plant physiology

involving high metabolic activity and generation of reac-

tive oxygen species (ROS) in the cell [1]. ROS affect

various aspects of seed physiology, displaying two major

functions: as a kind of cytotoxin and as a special role in

seed development, dormancy breakage, and in defence

against biotic and abiotic stresses [2]. A series of new

roles for ROS has recently been identified: the control

and regulation of biological processes, such as cell cycle,

programmed cell death and hormone signaling [3]. These

studies extend our understanding of ROSs and suggest a

dual role for ROS in plant biology as both toxic bypro-

ducts of aerobic metabolism and key regulators of

growth, development and defence pathways. ROS are

produced in aerobic organisms within the cell and are

normally in balance with antioxidant molecules. Oxida-

tive stress arises from an imbalance between generation

and elimination of ROS. These cytotoxic activated ROS

can seriously disrupt normal metabolism through oxida-

tive damage to lipids, protein and nucleic acids, selective

permeability of bio-membranes, causing membrane leak-

age and changes in the activity of membrane-bound en-

zymes [2]. However, an elaborate and highly redundant

plant ROS defence network, composed of antioxidant

enzymes, antioxidants and ROS-producing enzymes, is

responsible for maintaining ROS levels under tight con-

trol. In plant cells, antioxidant enzymes, such as super-

oxide dismutase (SOD), glutathione peroxidase (GPx),

peroxidase (POD), catalase (CAT) and ascorbate oxidase

(AO), are considered to form a defensive team, whose

combined purpose is to protect cells from oxidative da-

mage during growth, development and senescence [4].

Thus the key objective of this study was to determine

the impact of germination on biochemical and enzymatic

antioxidant activities of Ceiba pentandra seeds belong-

ing to the order Malvalea and the family Malvaceae.

2. Materials and Methods

2.1. Chemicals

Chemicals and reagents used for antioxidant estimations

were purchased from Merck. All additional chemicals used

were analytical grade. Altogether the experiments were

performed at room temperature unless otherwise stated.

2.2. Collection of Seeds

Mature Dried fruits of Ceiba pentandra were obtained

*Corresponding author.

Impact of Germination on Biochemical and Antioxidant Enzymes of Ceiba pentandra (Kapok) Seeds

1188

from in and around Andhra University area, Visakhapat-

nam, India. Healthy seeds were selected and washed

thoroughly with running tap water and with 5% (w/v)

Teepol for 10 minutes followed by treatment with ba-

vistin, a commercial fungicide for 5 minutes. Then the

seeds were subsequently surface sterilized with 0.1% (w/v)

HgCl2 for 5 minutes and then washed with sterile dis-

tilled water. The seeds were soaked for 24 hours before

they were kept for germination [5] in sterile Petri-plates

with double layered moistened filter paper. The germina-

tion was carried out at 30˚C with 16 hours light and 8

hours dark [6]. Radicle emergence of 1 cm was used as a

reference to consider seed germination. The 4 days ger-

minated seeds were used for biochemical and antioxidant

analysis.

2.3. Determination of Proteins, Amino Acids,

Total Soluble Sugars and Reducing Sugars

One gram of non-germinating (soaked overnight in 0.2 M

Tris HCl Buffer, pH 7.2) and germinating Ceiba pentan-

dra seeds were homogenized separately with 20 ml of

pre-chilled 0.2 M Tris HCl Buffer, pH 7.2, containing 0.1

mM EDTA in chilled pestle and mortar. The homoge-

nates were squeezed through double layered cheese cloth

and centrifuged (Sorvall Instrument RC5C, Rotor SS-34)

at 16,000 rpm for 15 minute at 4˚C. One ml of above ex-

tract was taken and 1 ml of ice cold 20% TCA was added

[7]. The pellet was washed twice with acetone and again

centrifuged at 8000 rpm. Supernatant was discarded and

pellet was dissolved in 5 mL of 0.1 N NaOH. This was

used for protein estimation. Total proteins were esti-

mated by the method of Lowry et al. [8] using BSA as

standard. Soluble sugars and free amino acids were de-

termined by phenol sulphuric acid method using glucose

as standard [9] and ninhydrin method using leucine as

standard [10].

2.4. Assay of Superoxide Dismutase

The assay of superoxide dismutase was carried out based

on the reduction of Nitroblue tetrazolium (NBT) [11]. To

0.5 ml of seed extract, 1 ml 125 mM of Sodium Carbon-

ate, 0.4 ml of 24 µM NBT and 0.2 ml of 0.1 mM EDTA

were added. The reaction was initiated by adding 0.4 ml

of 1mM Hydroxylamine hydrochloride. Zero time absor-

bance was taken at 560 nm using spectrophotometer,

followed by recording the absorbance after 5 min at 25˚C.

The control was simultaneously run without seed extract.

Units of SOD were expressed as amount of enzyme re-

quired for inhibiting the reduction of NBT by 50%. The

specific activity was expressed in terms of Units per mg

of protein.

2.5. Assay of Catalase

Catalase activity was determined by the titrimetric me-

thod [12]. To 1 ml plant extract, 5 ml of 300 μM phos-

phate buffer (pH 6.8) containing 100 μM hydrogen per-

oxide (H2O2) was added and left at 25˚C for 1 min. The

reaction was arrested by adding 10 ml of 2% sulphuric

acid, and residual H2O2 was titrated with potassium per-

manganate (0.01 N) till pink colour was obtained. Units

of enzyme activity were expressed as ml of 0.1 N potas-

sium permanganate equivalents of H2O2 decomposed per

mg protein per min.

2.6. Assay of Peroxidase

Assay of Peroxidase activity is carried out according to

the procedure of Malik and Singh [13]. 3.5 ml of phos-

phate buffer (pH 6.5) was taken in a clean dry cuvette,

0.2 ml seed extract and 0.1 ml of freshly prepared o-dia-

nisidine solution was added. The temperature of assay

mixture was brought to 28˚C - 30˚C and then placed the

cuvett in the spectrophotometer set at 430 nm. Then, 0.2

ml of 0.2 M H2O2 was added and mixed. The initial ab-

sorbance was read and then, at every 30 sec intervals up

to 3 min. A graph was plotted with the increase in absor-

bance against time. From the linear phase, the change in

absorbance per min was read. Water blank was used in

the assay. The enzyme activity was expressed in units per

mg of protein per min.

2.7. Assay of Glutathione Peroxidase

Glutathione Peroxidase was assayed by the method of

Rotruck et al. [14]. 0.2 ml each of 0.8 mM EDTA, 10

mM sodium azide, 1 mM GSH, 2.5 mM H2O2, 0.32 M

phosphate buffer (pH 7.0) and seed extract were mixed

and incubated at 37˚C for 10 min. The reaction was ar-

rested by the addition of 0.5 ml of 10% TCA and the

tubes were centrifuged. To 0.5 ml of supernatant, 3.0 ml

of 0.33 mM phosphate solution and 1.0 ml 0.6 mM

DTNB reagent were added and the colour developed was

read at 420 nm immediately. Graded amount of standards

were also treated similarly. Glutathione peroxidase acti-

vity was expressed as µg of glutathione utilized per mg

of protein.

2.8. Assay of Ascorbate Oxidase

To 3.0 ml of ascorbate solution (0.003%), 0.1 ml of plant

extracts were added and change in absorbance at 265 nm

was measured at an interval of 30 s for a period of 5 min.

One unit of enzyme activity was expressed as 0.01 OD

change per mg of protein [15].

3. Statistical Analysis

The results of in vitro study were given as Mean ± Stan-

Impact of Germination on Biochemical and Antioxidant Enzymes of Ceiba pentandra (Kapok) Seeds 1189

dard Deviation (SD) obtained from three independent ex-

periments, and analyzed with Student’s t-test for paired

data and a “p” value less than 0.05 was considered as

significant difference in the analysis.

4. Results and Discussion

4.1. Biochemical Components

The total protein content decreased during seed germina-

tion in ceiba pentandra seeds. The total protein content

in non-germinating seeds was 27 mg/gram tissue and

decreased to 11.6 mg/g tissue in four days germinating

seeds. On the contrary it was observed that there was an

increase in the free amino acids from 2.20 mg/g of tissue

in germinating seedlings to 0.78 mg/g of tissue in non-

germinating seeds. It may be due to rapid hydrolysis of

proteins, which would result in the release of free amino

acids. Similar fallouts were reported by Ali Al-Heal [16]

in Cassia senna seedlings. Considerable decrease in the

protein content was observed in germinating Lupinus

luteus and L. angustifolius [17] soybeans [18], Bambara

groundnuts (Voandzeia subterranea L. Thouans) [19],

fluted pumpkin (Telfairia occidentalis Hook) [20], and

sunflower seeds (Helianthus annuus) [21]. The total

soluble sugars content varied from 4 mg/g of tissue to 1.4

mg/g of tissue and reducing sugars varied from 0.56

mg/g of tissue and 1.26 mg/g of tissue in non-germina-

ting and germinating seeds respectively. During germi-

nation, there was a decrease in storage carbohydrates and

an increase of reducing sugars. This might be due to re-

quirement of energy by growing plant at initial stages of

seed germination. The outcomes acquired were presented

in Figure 1(a). These results agree well with the results

of Jaya and Venkataraman [22] in chickpea and green

gram and also in white beans by Kon et al. [23].

4.2. Antioxidant Activity

The germination process modified the antioxidant acti-

vity; after a germination period of four days, Ceiba pen-

tandra seeds showed higher antioxidant activity than raw

seeds. Capacity measured by the constraints of enzymatic

antioxidants includes SOD, CAT, POD, GPx and AO, all

the activities increased with increasing concentrations

ranging from 25 to 100 mg/ml. In germinating seed ex-

tract higher enzymatic antioxidants at 100 mg/ml like

SOD by 6.3 ± 0.089, CAT with 21.1 ± 0.089, peroxidase

69.2 ± 0.15, ascorbate oxidase through 0.84 ± 0.0009 and

glutathione peroxidase with 836 ± 0.89 were detected

against non-germinating seed extracts specifically SOD

with 1.99 ± 0.005, CAT with 6.77 ± 0.05, peroxidase

with 0.127 ± 0.0009, ascorbate oxidase with 0.29 ±

0.0005 and glutathione peroxidase with 84 ± 0.36.

The control of steady-state ROS levels by SOD is an

important protective mechanism against cellular oxida-

tive damage, since 2



acts as a precursor of more cyto-

toxic or highly relative ROS [24]. SOD has been estab-

lished to work in collaboration with POD and CAT

which act in tandem to remove 2 and H2O2, respec-

tively [4]. Early reports illustrate that increased SOD

activities and cellular ROS levels were involved in the

life of many plants including developmental course such

as seed germination [25]. Enhanced SOD activity can be

triggered by increased production of ROS or it might be

a protective measure adopted by C. pentandra seeds

against oxidative damage. Moreover, the changes of

SOD activity in the degrading endosperms and develop-

ing cotyledons were correlated to those of POD and CAT

activities. Our findings, shown in Figure 1(b) were also

in line with previous reports symptomatic of the partici-

pation of SOD in the defense mechanism during germi-

nation and early seedlings development [26].

O

In oily seeds, CAT is particularly important in the

early events of seedling growth, because it removes H2O2

produced during β-oxidation of the fatty acids [1]. In the

present study, CAT activity was also examined, and a

trend parallel to above was recorded; results are illus-

trated in Figure 1 (c). Increased CAT activity could be an

indication of the cellular evaluated ROS, since the

amount of CAT present in aerobic cells is directly pro-

portional to the oxidative state of the cells [2].

In plants, GPx and POD were considered to be associ-

ated with a number of essential metabolic processes, such

as cell elongation, lignification, phenolic oxidation, patho-

gen defense and defense against stress [27,28]. Moreover,

PODs probably play an important role in seed germina-

tion, growth, morphogenesis, and even in the final stage

of senescence and death [29,30]. Changes in PODs ac-

tivities occur during developmental process in tissue spe-

cific manner and differential regulation in response to

germination process and plant species has been reported

[31,32]. The present results indicate that PODs activities

in the degrading endosperms and developing cotyledons

were considerably greater than those of the raw seeds.

Outcomes were laid on sight in Figures 1(d) and (e).

Thus, increased PODs activities might be involved in the

defence system during seed germination and early seed-

lings development.

AO catalyses the oxidation of L-AA (L-Ascobic acid)

to MDHA (Monodehydroascorbate reductase) with the

related reduction of molecular oxygen to water [33]. In-

creasing evidence links this enzyme to the modulation of

cell expansion and/or cell division, possibly via control

of the oxidation status of the L-AA/DHA redox pair [34,

35]. It is highly expressed in fast-growing tissues [36]

and germinating pea seeds similar to that of above anti-

oxidant enzymes. AO is also found localised mainly in

the cell wall and AO mRNA and proteins are highly ex-

pressed in flowers, ovaries and very young fruits as well

Impact of Germination on Biochemical and Antioxidant Enzymes of Ceiba pentandra (Kapok) Seeds

1190

Figure 1. Levels of biochemical and antioxidant enzymes of Ceiba pentandra (kapok) seeds (a) Biochemical components; (b)

SOD; (c) Catalase; (d) Glutathione peroxidase; (e) Peroxidase; (f) Ascorbate oxidase (values represent average of triplicates

and expressed as mean ± SD).

as in the outer portions of the melon fruit mesocarp [37].

A similar expression pattern has also been found in non-

cucurbitaceous plants [38]. Thus AO-mediated oxidation

of apoplastic L-AA appears to be closely linked to cell

elongation processes. Our outcome was also in row with

preceding reports and those were put on view in Figure

1(f).

5. Conclusion

The seeds of Ceiba pentandra were rich in proteins and

carbohydrates, their levels decrease with the germination

progress, indicating their key role in the growth of em-

bryonic axis. Moreover, our findings strongly support the

hypothesis that SOD, POD, CAT and AO activities are

up-regulated as an antioxidant defense system against

endogenous oxidant radicals generated during seed ger-

mination. The whole biological consequences of these

alterations, in particular, low molecular mass antioxi-

dants as well as altered antioxidant defense mechanisms

during seed germination, are unclear and should be fur-

ther investigated.

6. Acknowledgements

I Ch. Ravi Kiran, duly acknowledge the financial assis-

Impact of Germination on Biochemical and Antioxidant Enzymes of Ceiba pentandra (Kapok) Seeds 1191

tance from University Grants Commission, NON-SAP,

New Delhi without which this project would never been

materialized.

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